Anti caspase 11

Rats (n=5 in each group) were sacrificed 72 h after SCI, and protein homogenates were prepared from samples incubated in pyrolysis liquid for 2 h. The samples were centrifuged at 4°C and 12 000 rpm for 30 min, and the supernatant was collected and stored separately. The protein concentration of the sample supernatant was measured with a BCA kit (Beyotime Biotechnology). Total protein (60 μg) separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis was transferred to a polyvinylidene fluoride membrane (Beyotime Biotechnology). The membrane was blocked with 5% skim milk powder at room temperature for 2 h and incubated overnight with a primary antibody at 4°C. The primary antibodies included anti-P2X7 (1: 2000, Thermo Fisher Scientific), anti-NLRP1 (1: 1000, Abcam), anti-NLRP3 (1: 2000, Abcam), anti-XIAP (1: 1000, Cell Signaling Technology), anti-ASC (1: 2000, Novus Biologicals), anti-caspase-11 (1: 2000, Cell Signaling Technology), anti-caspase-1 (1: 2000, Abcam), and anti-β-actin (1: 5000, Proteintech Group) antibodies. Then, the membranes were washed with Tris-buffered saline containing Tween and incubated with secondary antibodies (1: 5000, Abcam) at room temperature for 2 h. Subsequently, protein bands were visualized with a chemiluminescence kit (Beyotime Biotechnology, Beijing), and images were collected using sensitive film and chemical imaging software.

The total proteins were extracted using RIPA lysis buffer (Abcam, Beijing, China). Protein concentrations were measured using BCA Protein Assay Kit (Abcam); 20 μg proteins were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and then transferred to polyvinylidene difluoride membranes. After blocking, membranes were incubated with primary antibodies for overnight at 4°C. Next day, membranes were washed and blotted with horseradish peroxidase–conjugated secondary antibodies. Immunoreactive bands were detected using ECL Substrate Kit (Abcam). Primary antibodies used in present study were listed as follows: anti-caspase-11 (#14340; Cell Signaling Technology, Danvers, MA), Anti-GSDMD (#ab209845; Abcam), Anti-GSDMD-N (SAB1307178; Sigma-Aldrich, St. Louis, MO), anti-Pro-IL-1β (#12242; Cell Signaling Technology), anti-cleaved IL-1β (#52718; Cell Signaling Technology), and anti-β-actin (Proteintech, Wuhan, China). The band intensity was quantitated and analyzed by ImageJ (Version 1.8.0_172, National Institutes of Health, Bethesda, MD).